The adult mouse retina samples were obtained from a pool of C57/B6 mice (n = 350), which were purchased from Charles River Laboratories (Charles River Laboratories, Inc., Willmington, MA). All samples were placed in TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at -80ºC before RNA extraction. Total RNA was extracted with TRIzol reagent according to the manufacturer's instructions. For a quality control measure of the samples, total RNA was ran through a 1% agarose gel and using the 2100 Agilent BioAnalyzer System (Agilent Technologies, Palo Alto, CA) to check for possible contamination and degradation.
The mouse cortex samples were obtained from P1 C57/B6 mice (n = 19), which were purchased from Charles River Laboratories (Charles River Laboratories, Inc., Willmington, MA).The mouse cortex was used as a reference sample. Universal mRNA reference was still being developed when the study started, so it was not utilized. All samples were placed in TRIzol reagent (Invitrogen, Carlsbad, CA) and stored at -80ºC before RNA extraction. Total RNA was extracted with TRIzol reagent according to the manufacturer's instructions. For a quality control measure of the samples, total RNA was ran through a 1% agarose gel and using the 2100 Agilent BioAnalyzer System (Agilent Technologies, Palo Alto, CA) to check for possible contamination and degradation.
Extracted molecule
total RNA
Label
Cy3
Hybridization protocol
cDNA microarrays were constructed at the BioDiscovery Center at Biogen Idec (Cambridge, MA). RNA samples were reverse transcribed and cDNAs were amplified for 16-20 cycles using the SMART system (Clontech). Previous studies have shown that expression data generated by this approach are comparable to results obtained by other results (ref). To generate probes for array hybridization, 10ug cDNA was labeled by incorporation of either Cy5 or Cy3-dCTP (Amersham-Pharmacia) during oligo-dT-primed or random hexamer-primed primer extension in the presence of Klenow DNA polymerase (Roche). Arrays were pre-hybridized with poly-adenylic acid (Sigma) and mouse Cot-1 DNA (Life Technologies) before overnight hybridization with paired Cy3 and Cy5-labeled probes in the presence of poly-adenylic acid and mouse Cot-1 DNA. Pairs of labeled probes were hybridized at 42 overnight. Arrays were washed at room temperature in 0.2 × SSC, 0.1%SDS for 2 min, followed by 2 washes in 0.2 × SSC for 2 min each.
